Biomarkers can provide for early stage detection of a wide variety of diseases. As such, there is an urgent need to discover novel biomarkers that provide sensitive and specific disease detection (Aebersold et al., Proteome Res 2005, 4, (4), 1104-9; Srinivas et al., Clin Chem 2002, 48, (8), 1160-9). Biomarkers provide a way to diagnose a disease before clinical pathologies appear, allowing for early stage treatment of the disease, which typically provides better results.
For example, cancer is rapidly becoming the leading cause of death for many population groups in the United States, largely due to the fact that various types of the disease are usually diagnosed after the cancer has metastasized. At this later stage of the disease, treatment is typically invasive and ineffective. It is widely believed that early detection of cancer prior to metastasis will lead to a dramatic improvement in treatment outcome.
Biomarkers are also continually being discovered that are indicative of various other disease states and conditions as varied as Alzheimer's disease and diabetes. For many of these diseases, the early diagnosis of the disease allows for treatment options that have a greater chance of success than late stage treatment. Further, in some cases, early diagnosis of a disease or predisposition to a disease may even allow the person diagnosed to make lifestyle changes that may help to prevent and reverse the course of the disease without the need for more involved medical treatment.
Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of disease (Frank and Hargreaves; Nature reviews 2003, 2, (7), 566-80). Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole (Esp in a et ai. Proteomics 2003, 3, (11), 2091-100). Two major hurdles have prevented these discoveries from reaching clinical benefit: 1) disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture of biomolecules and could be masked by high-abundance species such as albumin, and 2) degradation of protein biomarkers can occur immediately following the collection of blood or body fluid as a result of endogenous or exogenous proteinases.
The concentration of proteins and peptides comprising the complex circulatory proteome ranges from 10-12 mg/mL to 10-3 mg/mL, spanning ten orders of magnitude, with a few high molecular weight proteins such as albumin and immunoglobulins accounting for 90% of total protein content (Anderson and Anderson, Mol Cell Proteomics 2002, 1, (11), 845-67). However, the low abundance and low molecular weight proteins and metabolites also present in the blood provide a wealth of information and have great promise as a source of new biomarkers. Conventional methods, such as two dimensional gel electrophoresis, do not have the sensitivity and resolution to detect and quantify low abundance low molecular weight proteins and metabolites. Also, in spite of the moderately high sensitivity of modem mass spectrometers (attomolar concentration), their working range spans over three-four orders of magnitude and therefore the less abundant proteins are masked by more abundant proteins. Consequently, the usual sample preparation steps for mass spectrometry (MS) experiments begin with depletion of high abundant proteins using commercially available immunoaffinity depletion columns (Agilent, Sigma, and Beckman-Coulter). After depletion, fractionation is performed by means of size exclusion chromatography, ion exchange chromatography, and/or isoelectric focusing. However, removal of abundant native high molecular weight proteins can significantly reduce the yield of candidate biomarkers because it has been recently shown that the vast majority oflow abundance biomarkers are non-covalently and endogenously associated with the carrier proteins that are being removed (Lopez et al., Clinical chemistry 2007, 53, (6), 1067-74; Conrads et al., BioTechniques 2006, 40, (6), 799-805; Lowenthal et al., Clin Chem 2005, 51, (10), 1933-45; Lopez et al., Clinical chemistry 2005, 51, (10), 1946-54). Methods, such as size exclusion ultrafiltration under denaturing conditions (Zolotarjova et al., Proteomics 2005, 5, (13), 3304-13), continuous elution denaturing electrophoresis (Camerini et al., Proteomics Clin. Appl. 2007, 1, 176-184), or fractionation of serum by means of nanoporous substrates (Geho et al., Bioconjug Chem 2006, 17, (3), 654-61) have been proposed to solve this problem. Moreover, these same recent findings point to the low molecular weight region of the proteome, as a rich and untapped source of biomarker candidates (Tirumalai et al., Molecular & cellular proteomics 2003, 2, (10), 1096-103; Merrell et al., J of biomolecular techniques 2004, 15, (4), 238-48; Orvisky et al., Proteomics 2006, 6, (9), 2895-902).
In addition to the difficulties associated with the harvest and enrichment of candidate biomarkers from complex natural protein mixtures (such as blood), the stability of these potential biomarkers poses a challenge. Immediately following blood procurement (e.g. by venipuncture) proteins in the serum become susceptible to degradation by endogenous proteases or exogenous environmental proteases, such as proteases associated with the blood clotting process, enzymes shed from blood cells, or associated with bacterial contaminants. Therefore, candidate diagnostic biomarkers in the blood may be subjected to degradation during transportation and storage. This becomes an even more important issue for the fidelity of biomarkers within large repositories of serum and body fluids that are collected from a variety of institutions and locations where samples may be shipped without freezing.
As such, there is a need in the art for particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures of biomolecules such as plasma, and protect them from degradation during subsequent sample handling. The captured analytes could then be readily extracted from the particles by electrophoresis allowing for subsequent quantitative analysis. Particles of this type would provide a powerful tool that is uniquely suited for the discovery of novel biomarkers for early stage diseases such as cancer.
Use of harvesting nanoparticles as created in a laboratory by the inventors of the present invention also has been shown to capture, protect from degradation, and amplifY the concentration of low abundance biomarkers in the urine. Human growth hormone within urine at low undetectable concentrations was concentrated by particle sequestration to be readily measured by a standard clinical grade immunoassay. For the first time this labile and low abundance biomarker can now be routinely screened in the urine. Physiologic salt and urea concentration does not affect the function of the particle sequestration. The captured biomarker is preserved and stable at room temperature or at 37C. This finding is applicable to any desired biomarker that can be captured by the particles and uniquely solves a need.